A solid-phase synthesis method which uses the phosphoramidite method is broadly used in the chemical synthesis of nucleic acids such as a DNA oligonucleotide and RNA. In this method, for example, a nucleoside which becomes the 3′-terminal of the nucleic acid to be synthesized is firstly supported in advance on a solid-phase synthesis support via a cleavable linker such as succinyl group, and this support is put into a reaction column and set on an automatic nucleic acid synthesizer. Thereafter, synthesizing reagents are fed into the reaction column, for example, in the following manner in accordance with the synthesizing program of the automatic nucleic acid synthesizer. (1) Deprotection of nucleoside 5′-OH group by a trichloroacetic acid/dichloromethane solution, dichloroacetic acid/toluene solution or the like, (2) coupling reaction of amidite with the 5′-OH group by a nucleoside phosphoramidite (nucleic acid monomer)/acetonitrile solution and an activator (tetrazole or the like)/acetonitrile solution, (3) capping of the unreacted 5′-OH group by acetic anhydride/pyridine/methyl imidazole/THF or the like, and (4) oxidation of phosphite by iodine/water/pyridine or the like.
By repeating this synthesis cycle, a nucleic acid having the intended sequence is synthesized. The finally synthesized nucleic acid cut out from the solid-phase synthesis support by hydrolyzing the cleavable linker with ammonia, methylamine or the like (cf. Non-patent Reference 1).
As the solid-phase synthesis support to be used in the synthesis of nucleic acid, CPG (Controlled Pore Glass), silica gel and the like inorganic particles have so far been used, but in recent years, resin beads which can increase quantity of nucleic acid synthesized per weight of solid-phase synthesis support have been started to be used in order to synthesize at a moderate price and in a large quantity. As such resin beads, a highly-crosslinked and non-swelling porous polystyrene bead (cf. Patent Reference 1), a low-crosslinked and swelling porous polystyrene bead (cf. Patent Reference 2) and the like can for example be mentioned.
However, when supporting amount of the nucleoside linker as the starting point of synthesis is increased to too high level in order to further increase the quantity of nucleic acid synthesized per weight of solid-phase synthesis support, it poses a problem in that the coupling efficiency of amidite becomes poor and purity of the nucleic acid obtained is considerably lowered. For example, upper limit of the nucleoside linker supporting amount of a commercially available solid-phase synthesis support, which can synthesize a DNA oligonucleotide of 20 bases in high purity, is so far about 200 μmol/g at the most.
An attempt has been made to improve nucleic acid synthesizing ability by, using acrylonitrile, suppressing a fluctuation of swelling ratio of a porous resin bead in various organic solvents (cf. Patent Reference 3). However, although the porous resin bead used in this reference is small in the fluctuation of swelling ratio of the porous resin bead in various organic solvents, further improvement is in demand regarding the nucleic acid synthesizing ability.
Patent Reference 1: JP-A-3-68593
Patent Reference 2: JP-A-2005-325272
Patent Reference 3: JP-A-2008-74979
Non-patent Reference 1: Current Protocols in Nucleic Acid Chemistry (2000), UNIT 3.6 Synthesis of Unmodified Oligonucleotides